Name: an engulfment assay: a protocol to assess interactions between cns phagocytes and neurons
Uploaded: 2014-06-08T23:00:00
Description: Watch this Scientific Journal Video about An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons at JoVE.com

，NRSA（F32-NS-066698; DPS）;工作是由史密斯家庭基金会（BS），达纳基金会（BS），约翰·默克学者奖励计划（BS），NINDS（BS RO1-NS-07100801）资助项目，南希·劳瑞商标基金会（DPS），美国国立卫生研究院（P30-HD-18655; MRDDRC影像核心）。

1，顺行研资局突触前输入的标签注意：所有涉及使用动物实验，根据美国国立卫生研究院的所有准则审阅及机构动物护理和使用委员会（IACUC）监督。 <ol><li>消毒领域和手段。 </li><li>麻醉用鼠标在一个树脂玻璃感应室4％（体积）的异氟醚（这个体积％异氟醚适用于新生儿，成年小鼠）。观察小鼠的密切合作，以避免过度麻醉。注意：避免吸入用真空废气撤离。 </li><li>经过...

<ol>
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为了准确地测量吞噬作用，吞噬材料必须贴在这样一种方式，研究人员可以直观一次溶酶体降解发生。此外，高分辨率成像是必需的，随后使用软件，使研究者能够可视化整个细胞的体积和量化其内容。在这个协议中，我们描述了用于使用CTB偶联至Alexa染料来标记吞噬材料结合高分辨率共焦显微镜和三维重建测定吞噬细胞介导的​​吞噬一个高度可靠的和定量的方法。这种方法已被用来在实验室?...

<html>突触电路改造整个动物的生命。在脑发育，突触形成过剩，而且必须经过突触的修剪涉及选择性去除突触的一个子集和那些仍然8-10突触的维持和加强。这个过程是必需的，以实现对成人神经系统的精确的连接特性。在成人中，突触也可以是塑料，特别是在学习和记忆的上下文中。此可塑性的结构相互关系被认为是包括树突棘和突触前boutons 11-13的加法和/或消除。除了 ​​在健康的神经系统这些角色，突触重塑也参与了神经系统疾病/伤害12,14,15。例如，下面的脊髓损伤，轴突切断后必须改造，形成新的突触，实现功能的恢复16-19。 NT“&gt;新兴突触可塑性的一个重要方面是吞噬或拟用于去除3,5,20突触吞噬的过程中，我们最近发现这种现象在健康，出生后小鼠大脑突触7修剪的情况下，特别，小胶质细胞，常住中枢神经系统的免疫细胞和吞噬细胞，被证明在高峰期和发育突触的修剪，丘脑的产后背外侧膝状体核（外膝体）的区域吞没突触前输入。这吞没的遗传或药理封锁导致突触连接持续的赤字。 在这个协议中，我们描述了一种可靠的和高度的定量测定法来测量的突触前输入，吞噬细胞介导的​​吞噬。对于这篇文章的目的，此法将在发展中retinogeniculate系统，其中包括视网膜神经节细胞（RGC的）居住在视网膜的背景下提出的项目突触前输入到外膝体（ 图1A）。开始，溶酶体降解抗性顺行标记策略进行说明，这是用于可视化的外膝体RGC特异突触前输入（ 图1）7,21。以下本说明书中，用于成像的详细方法和使用共聚焦显微镜并结合3维（3D）表面体绘制的定量测定吞噬将得到。此方法是基于固定的组织制备，但也可以适于在实时成像研究使用。重要的是，尽管已经测定在健康，产后retinogeniculate系统的上下文中得到证实，人们可以应用相同的技术来评估其它吞噬细胞 - 神经元相互作用的整个大脑和疾病过程中，以及在其他器官系统的吞噬细胞的功能。

<table border="0" cellpadding="0" cellspacing="0" width="533">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:144px;">Heat pad</td>
			<td style="width:159px;">Vet Equip, Inc.</td>
			<td style="width:168px;">965500&#160;</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:144px;">Warm water source for heat pad</td>
			<td style="width:159px;">Kent Scientific</td>
			<td style="width:168px;">TP-700</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:144px;">Stereo microscope</td>
			<td style="width:159px;">DSC Optical</td>
			<td style="width:168px;">Zeiss Opmi -6 Surgical Microscope</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="40" rowspan="2" style="height:40px;width:144px;">Sliding microtome with freezing stage</td>
			<td rowspan="2" style="width:159px;">Leica</td>
			<td rowspan="2" style="width:168px;">SM2010 R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:144px;">Microtome blade</td>
			<td style="width:159px;">Leica</td>
			<td style="width:168px;">14021607100</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:144px;">Fluorescent dissecting microscope</td>
			<td style="width:159px;">Nikon</td>
			<td style="width:168px;">SMZ800 with Epi-fluorescence attachment</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:144px;">Spinning disk confocal microscope</td>
			<td style="width:159px;">Perkin Elmer</td>
			<td style="width:168px;">UltraView Vox Spinning Disk Confocal</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:131px;">10 &#181;l Hamilton gas tight syringes</td>
			<td style="width:131px;">Hamilton</td>
			<td style="width:116px;">80030</td>
			<td style="width:144px;">Use a different syringe for each color dye/tracer</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:131px;">Hamilton needles</td>
			<td style="width:131px;">Hamilton</td>
			<td style="width:116px;">7803-05, specifications: blunt, 1.5&#34;</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="20">
			<td height="54" rowspan="3" style="height:54px;width:131px;">Alexa-conjugated cholera toxin &#946; subunit (CTB)</td>
			<td rowspan="3" style="width:131px;">Invitrogen</td>
			<td style="width:116px;">488: C22841</td>
			<td rowspan="3" style="width:144px;">Reconstitute in sterile saline, 80 &#181;l (488), 100 &#181;l (594), 20 &#181;l (647)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:116px;">594: C22842</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:116px;">647: C34778</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Phosphate Buffered Saline&#160;(PBS)</td>
			<td style="width:131px;">Sigma</td>
			<td>P4417-50TAB</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:131px;">Neomycin and Polymyxin B Sulfates and Bacitracin Zinc Ophthalmic Ointment USP (antibiotic ointment)</td>
			<td style="width:131px;">Bausch &#38; Lomb</td>
			<td style="width:116px;">24208-780-55</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">30.5 gauge needle</td>
			<td style="width:131px;">Becton Dickinson</td>
			<td style="width:116px;">305106</td>
			<td rowspan="3" style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">Spring scissors</td>
			<td style="width:131px;">Roboz</td>
			<td style="width:116px;">RS-5630</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Cotton-tipped applicator</td>
			<td style="width:131px;">Fisher</td>
			<td style="width:116px;">23-400-125</td>
		</tr>
		<tr height="21">
			<td height="104" rowspan="2" style="height:104px;width:131px;">Paraformaldeyde (PFA)</td>
			<td rowspan="2" style="width:131px;">Electron Microscopy Sciences</td>
			<td rowspan="2" style="width:116px;">15710</td>
			<td rowspan="2" style="width:144px;">Dilute 16%to 4% in PBS. Paraformaldehye is toxic, use&#160; in a fume hood and wear personal protective equipment.</td>
		</tr>
		<tr height="83">
		</tr>
		<tr height="36">
			<td height="138" rowspan="4" style="height:138px;width:131px;">Dissection tools &#8211; scissors, forceps, spatula</td>
			<td style="width:131px;">Small scissors: Fine Science Tools</td>
			<td style="width:116px;">Small scissors:14370-22</td>
			<td rowspan="4" style="width:144px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Large scissors: Roboz</td>
			<td style="width:116px;">Large scissors: RS-6820</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">#55 forceps: Fine Science Tools</td>
			<td style="width:116px;">#55 forceps: 11255-20</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Spatula: Ted Pella, Inc.</td>
			<td style="width:116px;">Spatula: 13504</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:131px;">Sucrose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:116px;">S8501-5KG</td>
			<td style="width:144px;">Make 30% sucrose in PBS (weight/vol)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">OCT Compound</td>
			<td style="width:131px;">VWR</td>
			<td style="width:116px;">25608-930</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">Weigh boat</td>
			<td style="width:131px;">USA Scientific</td>
			<td style="width:116px;">2347-1426</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">24-well plates</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:116px;">353047</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">Sodium phosphate monobasic</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:116px;">S6566-500G</td>
			<td rowspan="2" style="width:144px;">Make 0.2 M sodium phosphate monobasic (PB-A) in ddH20 and 0.2 M sodium phosphate dibasic (PB-B) in ddH20.&#160; To make 0.1 M PB, combine 19 ml PB-A and 81 ml PB-B, fill to 200 ml with ddH20&#160;&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:131px;">Sodium phosphate dibasic&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td>S5136-500G</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Coverslips, 22 X 50 mm, No. 1.5</td>
			<td style="width:131px;">VWR</td>
			<td style="width:116px;">48393 194</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Charged microscope slide</td>
			<td style="width:131px;">VWR</td>
			<td style="width:116px;">48311-703</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">Vectasheild</td>
			<td style="width:131px;">Vector Laboratories</td>
			<td style="width:116px;">H-1200</td>
			<td style="width:144px;"></td>
		</tr>
	</tbody>
</table>

an engulfment assay: a protocol to assess interactions between cns phagocytes and neurons

吞噬作用是一个过程，其中一个细胞吞噬材料（整个小区的小区，杂物等部件）在其周围的细胞外环境，并随后消化此材料，通常通过溶酶体降解。小胶质细胞是中枢神经系统（CNS）的细胞的吞噬功能的广泛的神经退行性疾病（ 例如 ，β-淀粉样蛋白的间隙在阿尔茨海默氏病）的健康脑的发展（ 例如，突触条件已经描述的驻地免疫细胞修剪）1-6。下面的协议是制定可视化和量化，在发育中的小鼠retinogeniculate系统7的突触前输入，小神经胶质细胞介导的吞噬的吞噬测定。虽然这种测定法用于评估在此特定情况下的小胶质细胞的功能，类似的方法可用于评估其他吞噬细胞在整个脑（ 例如，星形胶质细胞）和身体的其余部分（ 例如 ，外周血巨噬细胞）以及在其中突触重塑发生（ 例如 ，脑损伤/疾病）其他上下文中。

最近，我们使用这个吞噬试验，以可视化和量化的显影retinogeniculate系统的突触前输入，小神经胶质细胞介导的吞噬（ 图1）7。从CX3CR1-EGFP杂合子小鼠视网膜神经节细胞进行顺行追踪与CTB-594和CTB-647进入左眼和右眼分别。在此之后追踪，外膝体中EGFP阳性的小胶质细胞进行成像。这些图像随后的表面呈现为体积测量。 使用这种技术，我们发现，在外膝体（P5?...

Watch this Scientific Journal Video about An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons at JoVE.com

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

小胶质细胞是中枢神经系统（CNS）具有高容量，以在它们的胞外环境吞噬或吞噬物质的驻留的免疫细胞。这里，用于可视化和测量突触小部件的小神经胶质细胞介导的​​吞噬一个广泛适用的，可靠的和高度的定量测定法进行说明。

一个吞噬含量：一个协议，以评估之间的中枢神经系统吞噬细胞和神经元的相互作用

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

Research

JoVE Journal

Neuroscience

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

神经细胞集落形成细胞分析检测，以区分善意的神经祖细胞的神经干细胞

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

科研

神经科学

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

幼虫和成人测绘和增强子陷阱Flippase表达中的应用果蝇中枢神经系统

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

在培养的中枢神经系统的神经元树突棘形态分析

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

剪切载荷：一个有用​​的工具，用于检查视网膜神经细胞之间的缝隙连接示踪剂耦合程度

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

一种方法为Target和隔离气道支配感觉神经元的小鼠

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

一种方法来提高背根神经节的对齐和髓鞘

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

条纹分析来研究蛋白质底物使用游离海马神经元的吸引力或令人反感的活动

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

间接神经元的星形胶质细胞共培养试验：一个<em&gt;体外</em&gt;设置 - 为神经元的神经胶质细胞相互作用的详细调查

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

Overexpressing 突触前蛋白培养神经元突触囊泡再生的光学检测

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...